Hepatic thrombopoietin gene silencing reduces platelet count and breast cancer progression in transgenic MMTV-PyMT mice. Shirai T, Revenko AS, Tibbitts J, Ngo ATP, Mitrugno A, Healy LD, Johnson J, Tucker EI, Hinds MT, Coussens LM, McCarty OJT, Monia BP, Gruber
Our group hypothesized that a decrease in levels of hepatic thrombopoietin(THPO) would correlate to a delay in tumor progression and metastasis in mammary cancer mouse models. I helped develop a series of experiments, where we utilized female MMTV-PyMT transgenic mice to investigate the effect of murine THPO-ASO treatment on blood platelet count, plasma TPO levels, and progression of breast cancer. Hemizygous MMTV-PyMT mice spontaneously developed ultimately fatal metastatic breast cancer in mammary glands, with early microscopic mammary gland epithelial atypia beginning as early as 3 to 4 weeks of age. FVB/N wild type mice were used for establishment of methods and to verify that THPO-ASO reduced TPO levels and platelet count in this mouse strain. Hemizygous MMTV-PyMT mice that develop cancer were used as comparators and to also determine if platelet count reduction affects the progression of their cancers. Our findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.
Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo. Shirai CL, Ley JN, White BS, Kim S, Tibbitts J, Shao J, Ndonwi M, Wadugu B, Duncavage EJ, Okeyo-Owuor T, Liu T, Griffith M, McGrath S, Magrini V, Fulton RS, Fronick C, O'Laughlin M, Graubert TA, Walter MJ.
Heterozygous somatic mutations in the U2AF1 occur in ∼11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. Our group examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a transgenic mouse model. Mice expressing mutant U2AF1 display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support our hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.
Reduced levels of Hspa9 attenuate Stat5 activation in mouse B cells. Krysiak K, Tibbitts JF, Shao J, Liu T, Ndonwi M, Walter MJ.
HSPA9 is a gene located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies, including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS. To evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, our group generated a Hspa9 knockout mouse model. Hspa9(+/-) mice have normal basal hematopoiesis and do not develop MDS. Our group further reduced Hspa9 expression (<50%) using RNA interference and observed reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B lymphopoiesis in vivo. Knockdown of Hspa9 in an interleukin 7 (IL-7)-dependent mouse B-cell line reduced signal transducer and activator of transcription 5 (Stat5) phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B cells. Collectively, these data support our theory that Hspa9 plays a major role in B lymphopoiesis and Stat5 activation downstream of IL-7 signaling. Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice. Chen TH, Kambal A, Krysiak K, Walshauser MA, Raju G, Tibbitts JF, Walter MJ. Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when the p53 gene is mutated. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, our group knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow–transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.
Vulnerability of Mouse Cortical Neurons to Doxorubicin-Induced Apoptosis is Strain-Dependent and is Correlated with mRNAs Encoding Fas, Fas-Ligand, and Metalloproteinases. M. Wetzel, J. Tibbitts, G.A. Rosenberg and L.A. Cunningham. Cell surface death receptor-mediated neuronal apoptosis, which is a critical component of neurodegeneration, is modulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Doxorubicin (Dox) induces neuronal death by the activation of death receptor pathways. Our group demonstrated that Dox-induced neuronal apoptosis is regulated by the balance of MMP-3 and TIMP-3 in cortical cultures by examining whether inbred mice display strain-dependent vulnerability to Dox. We induced neuronal apoptosis with Dox in primary neuronal cultures established from cerebral cortices of embryonic day 15 mice. Using fluorescence activated cell sorting for neurons, we found that C57BL/6 cortical cultures exhibit a 28% greater neuronal death following Dox treatment than C57BL/10. Real-time PCR of unstimulated cultures revealed that C57BL/10 cortical cultures have reduced basal mRNA levels encoding the pro-apoptotic proteins: Fas, FasL, and TIMP-3, but increased levels of the anti-apoptotic molecule MMP-3 as compared to C57BL/6. Furthermore, C57BL/10 cultures treated with Dox displayed an enhanced induction of mRNA transcripts that encode anti-apoptotic MMPs. Our results show that C57BL/10 cortical cultures are more resistant to death receptor-mediated apoptotic cell death as compared to C57BL/6, and suggest that this difference is related to Fas, FasL, and MMP expression.
Past projects
Early Opening of the Blood-Brain Barrier in Stroke Associated with Gelatinase Activity and MT1- Expression. Y Yang, E. Estrada, J. Tibbitts, J. Thompson, W.L. Liu, G. Quintana, and G. Rosenberg. Vulnerability of Mouse Cortical Neurons to Doxorubicin-Induced Apoptosis is Strain-Dependent. Monica Wetzel, Justin Tibbitts, C. William Shuttleworth, Gary A. Rosenberg, Lee Anna Cunningham. Focal Ischemia Increases Expression of Timp-3, MMP-3, Fas Receptor, and Fas Ligand in the Mouse Striatum. Yi Yang, Eduardo Y. Estrada, Justin F. Tibbitts, Jeffrey F. Thompson, Wenlan Liu, Gary A. Rosenberg.
MMP-2, MT1-MMP and Furin Associated with the Early Opening of the Blood-Brain Barrier (BBB) in Reperfusion Ischemic Injury in Rat Brain. Yi Yang, Eduardo Y. Estrada, Justin F. Tibbitts, Jeffrey F. Thompson, Wenlan Liu, Gary A. Rosenberg. Involvement of Matrix Metalloproteinases (MMPs) Following an Experimental Model of Intracerebral Hemorrhage in Mouse. Mark Grossetete, Justin F. Tibbitts, Gary A. Rosenberg.